Development of a fast and reliable methodology for the determination of polyamines in urine by using a guard column as a low-resolution fractioning step prior to mass spectrometry. Comparison with flow injection-mass spectrometry analysis
Introduction
Nowadays, there is an increasing interest on the development of fast and reliable methodologies that allow the analysis of a great number of samples within the shortest time possible and with instrumental setups common in research laboratories [1], [2]. With the use of mass spectrometry (MS), it is possible to perform the analysis with high sensitivity and selectivity and, depending on the coupled technique, with shorter or longer times of analysis. The use of strategies where the sample is introduced directly into the mass spectrometer allows shorter times of analysis compared to those in which there is a previous separation step (chromatographic or electrophoretic). In this way, it is possible to perform high throughput analysis, a very important aspect in routine analysis and when there is a large number of samples to analyze [2]. However, the use of these non separative methodologies based on direct injection into the mass spectrometer presents several disadvantages, such as matrix effects due to the absence of a previous separation step and the presence of isobaric interferences that could hamper analyte quantification if no specific fragmentation patterns are present [1], [3]. These problems can be alleviated by the use of different sample treatment techniques, but, in many cases, imply extensive sample manipulation, specifically if the compounds are present at low concentrations in complex matrices [4], [5], [6], [7], [8].
Using guard columns is a widespread practice in analytical chemistry with different purposes, mainly as column protection [9], [10], [11] or as a sample clean-up step [12], [13]. It also has been proposed the use of guard columns as chromatographic columns in order to perform liquid chromatography (LC), with times of analysis from 7 to 14 min [14], [15], [16]. Recently, Couchman et al. [17] have proposed an ultra-fast LC-MS method using a guard column as the chromatographic one for the sub-minute analysis of therapeutic drugs in plasma samples, but it required the use of high performance pumps and double-needle autosamplers.
In the present work, we propose a fast and reliable method for the determination of polyamines and related compounds in urine samples. Two different options were evaluated, i) the use of flow injection analysis coupled to a mass spectrometer with an electrospray ion source and a triple quadrupole (FIA-MS/MS) and ii) the inclusion of a guard column as a low-resolution fractioning step prior to MS quantification (gC-FIA-MS/MS), in order to increase the sensitivity of the analysis. The final objective is the development of a method for screening purposes. A methodology including a chromatographic separation step (LC-MS/MS) is also proposed in order to confirm the results obtained with the screening one. This chromatographic method will be only used when required, that is, to analyze samples with altered concentrations of the compounds.
Polyamines and acetylated polyamines have long been associated with cancer [18] and elevated concentrations of these compounds have been found in urine of patients diagnosed with this disease [19], [20], [21]. Ornithine and gamma-amino butyric acid, also included in this study, are involved in the metabolic pathway of polyamines [18], [22]. Accordingly, the development of fast and reliable methodologies for their determination is important, especially using samples that do not involve invasive sampling procedures. Several methodologies have already been proposed for the determination of the aforementioned compounds in urine samples, mainly using a chromatographic separation step [21], [23], [24], [25], [26]. Other non-separative methodologies have also been proposed, but they involve laborious sample preparative steps, such as solid phase extraction or the use of a derivatization reaction [4], [5], [27]. To the best of our knowledge, this is the first time that this fast approach (gC-FIA-MS/MS) is applied to the determination of these compounds in urine samples.
Section snippets
Chemicals
Putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), N-acetylputrescine (N-AcPut), N1-acetylspermine (N-AcSpm), L-ornithine (Orn) and γ-aminobutyric acid (GABA) were supplied by Sigma-Aldrich (Steinheim, Germany). Isotopically labelled internal standards putrescine-2,2,3,3-d4 (Put-d4), spermidine-(butyl-13C4) (Spd-13C4) and 4-aminobutyric acid-2,2,3,3,4,4-d6 (GABA-d6) were also supplied by Sigma-Aldrich, as well as methanol, heptafluorobutyric acid (HFBA), trifluoroacetic acid
Urine sample preparation
Protein content is a critical parameter because of ion suppression, especially when the samples are injected directly into the mass spectrometer. Protein precipitation was accomplished by the use of an acidified organic medium with different volatile acids [24], [26]. HFBA, formic acid and acetic acid were tested (0.1%, v/v), using MeOH as organic medium. The precipitation medium (300 µL) was mixed with 250 µL of the thawed urine and the mixture was vortexed (5 min) and centrifuged at 1815g for
Conclusions
Here we have proposed a fast and reliable method for the determination of polyamines and related compounds in urine samples. The inclusion of a guard column in the FIA-MS/MS instrumental configuration resulted in a low-resolution fractioning of the target compounds with an increment on the analytical signals but without an increase on the time of analysis. All the steps involved in sample preparation and instrumental conditions were optimized. Urine samples were centrifuged and filtered using
CRediT authorship contribution statement
María Teresa Fernández-del-Campo-García: Investigation, Validation, Formal analysis, Writing - original draft. Ana María Casas-Ferreira: Conceptualization, Supervision, Writing - review & editing, Visualization. Encarnación Rodríguez-Gonzalo: Conceptualization, Supervision, Writing - review & editing, Visualization. Bernardo Moreno-Cordero: Writing - review & editing. José Luis Pérez-Pavón: Conceptualization, Writing - review & editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors wish to thank the Spanish Ministry of Economy and Competitiveness for funding project CTQ2017-87886-P/BQU and the Junta de Castilla y León for project SA055P17. Mª Teresa Fernández-del-Campo-García is also thankful to the University of Salamanca for a predoctoral fellowship.
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